Hydroxylysine Derivatives

In addition to the 20 common amino acids, proteins may contain residues created by modification of common residues already incorporated into a polypeptide (Fig. 3-8a). Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from lysine. The former is found in plant cell wall proteins, and both are found in collagen, a fibrous protein of connective tissues. 6-N-Methyllysine is a constituent of myosin, a contractile protein of muscle. Another important uncommon amino acid is y-carboxyglutamate, found in the bloodclotting protein prothrombin and in certain other proteins that bind Ca2+ as part of their biological function. More complex is desmosine, a derivative of four Lys residues, which is found in the fibrous protein elastin. 

Fig. 5. Synthesis of 1,2-trans glycosides using a participating group at C-2 for example, synthesis of glycosylated hydroxylysine derivative (p-D-Gal-( 1 >0)-IIyl) using (a) insoluble promoter-silver silicate (56) (b) soluble promoter-silver trifluo-romethanesulfonate (57).

Malkar, N B, Lauer-Fields, J L, Fields, G B, Convenient synthesis of glycosylated hydroxylysine derivatives for use in solid-phase peptide synthesis. Tetrahedron Lett., 41, 1137-1140, 2000. 

Marin J, Didierjean C, Aubry A, Casimir JR, Briand JP, Guichaid G (2004) Synthesis of enantiopure 4-hydroxypipecolate and 4-hydroxylysine derivatives from a commmi 4,6-dioxopiperidinecarboxylate precursor. J Org Chem 69 130-141... 

Considerable activity continues with 0-glycosyl amino-acids. Two reports have appeared on 3-D-galactopyranosyl L-hydroxylysine derivatives,the first in connection with the development of assays for osteoporosis. An extensive range of related compounds W (with R = alkyl and fluoroalkyl), as well as related thioglycoside and 2-N-acetyl-2-deoxy compounds have been made as GalCer analogues for anti-HIV testing. ... 

Humans can synthesize 12 of the 20 common amino acids from the amphiboHc intermediates of glycolysis and of the citric acid cycle (Table 28-1). While nutritionally nonessenrial, these 12 amino acids are not nonessential. AH 20 amino acids are biologically essential. Of the 12 nutritionally nonessential amino acids, nine are formed from amphibolic intermediates and three (cysteine, tyrosine and hydroxylysine) from nutritionally essential amino acids. Identification of the twelve amino acids that humans can synthesize rested primarily on data derived from feeding diets in which purified amino acids replaced protein. This chapter considers only the biosynthesis of the twelve amino acids that are synthesized in human tissues, not the other eight that are synthesized by plants. 

Hi. Lysine. Gamma radiolysis of aerated aqueous solution of lysine (94) has been shown, as inferred from iodometric measurements, to give rise to hydroperoxides in a similar yield to that observed for valine and leucine. However, attempts to isolate by HPLC the peroxidic derivatives using the post-column derivatization chemiluminescence detection approach were unsuccessful. This was assumed to be due to the instability of the lysine hydroperoxides under the conditions of HPLC analysis. Indirect evidence for the OH-mediated formation of hydroperoxides was provided by the isolation of four hydroxylated derivatives of lysine as 9-fluoromethyl chloroformate (FMOC) derivatives . Interestingly, NaBILj reduction of the irradiated lysine solutions before FMOC derivatization is accompanied by a notable increase in the yields of hydroxylysine isomers. Among the latter oxidized compounds, 3-hydroxy lysine was characterized by extensive H NMR and ESI-MS measurements whereas one diastereomer of 4-hydroxylysine and the two isomeric forms of 5-hydroxylysine were identified by comparison of their HPLC features as FMOC derivatives with those of authentic samples prepared by chemical synthesis. A reasonable mechanism for the formation of the four different hydroxylysines and, therefore, of related hydroperoxides 98-100, involves initial OH-mediated hydrogen abstraction followed by O2 addition to the carbon-centered radicals 95-97 thus formed and subsequent reduction of the resulting peroxyl radicals (equation 55). 

Leucine oxidized with "OH produces a 5 -hydroperoxy derivative which is subjected to chemical reduction to yield (2S)-y-hydroxyleucine, (2S,4S)-8-hydroxy-leucine, (2S,4R)-8-hydroxyleucine. The S -hydroxyleucines have been confirmed to be the reduction products of the corresponding hydroperoxyleucines. 5 -Hydro-xylysines are natural products formed by lysyl oxidase and therefore are not useful markers of radical-mediated oxidation. The other hydroxylysines are useful markers, however, with HPLC analysis of 9-fluorenylmethyl chloroformate (FMOC)... 

During detailed characterization of a tryptic map of recombinant human tissue plasminogen activator (rtPA), a minor peak was resolved whose mass was not consistent with the expected set of tryptic peptides (L. Keyt and S.-L. Wu, unpublished observation). N-terminal sequence analysis of this fraction showed that it had a sequence containing residues 276-296 of rtPA with Lys-277 present as Hyl. We developed a modified amino acid analysis program capable of detecting hydroxylysine at levels down to 0.05 residues/mol to determine the distribution of Hyl in rtPA as well as in some other proteins derived from mammalian cells. 

Recent studies on separation optimization showed that accurate control of mobile phase pH was essential to successfully resolve a number of important non-hydrolysate amino acids. With good control of a complex gradient profile the system could resolve a mixture of amino acids including, Asn, Gin, cysteine derivatives carboxymethyl cysteine and pyridylethyl cysteine, and the hydroxylated amino acids hydroxyproline (Hyp) and hydroxylysine (Hyl) as well as the hydrolysate amino acids (4). However, the required precision in the control of eluent pH unnecessarily complicated transfer of the method between laboratories. The method also lacked the ability to separate Orn from the hydrolysate amino acids. The current study demonstrates the utility of quaternary HPLC gradient systems for facilitating methods development and simplifying routine eluent preparation with excellent pH control. 

Several other types of covalent crosslinks, mostly derived from lysine or 5-hydroxylysine residues (the latter being formed by post-translational modification), are found in collagen and elastin. A few examples are given (5.2-5.7) A6 7-dehy-drolysinonorleucine (5.2), lysinonorleucine (5.3), dehydrohydroxylysinonorleucine (5.4), lysino-5-ketonorleucine (5.5), desmosine (5.6) and isodesmosine (5.7). An intrachain thiol ester loop is present in a2-macroglobulin and proteins of the complement system and consists of a fifteen-membered ring derived from cysteine and glutamic acid (5.8). 

The tropocollagen [5- and y-chains in Fig. 4.1 are respectively twice and three-times the size of the a-chain. Indeed, their amino acid composition is virtually identical to the a-chain in Fig. 4.1, except for the appearance of derivatives of lysine and hydroxylysine, mainly lysyl hydroxyl-norleucine and pyridinoline. They increase in amount with age of the organism. 

The oligosaccharide is involved at its reducing end in a glycosidic bond with one of the hydroxylated side chain residues of the polypeptide. They are the ones derived from L-serine 13.11, L-threonine 13.12, L-hydroxylysine 13.13, and... 

All the fibril-forming collagen t) es (t) e I, II, III, V, XI, XXIV, and XXVII collagens) are cross-linked through a mechanism based on the reactions of aldehydes derived from some lysine (or hydroxylysine) side chains. Histidine might also participate in the formation of a trivalent cross-link by reacting with an aldimine bond formed between a lysine aldehyde and hydroxylysine residue. 

Many other amino acids, in addition to the ones listed here, are known to exist. They occur in some, but by no means all, proteins. Figure 3.4 shows some examples of the many possibilities. They are derived from the common amino acids and are produced by modification of the parent amino acid after the protein is synthesized by the organism in a process called posttranslational modification. Hydroxyproline and hydroxylysine differ Ifom the parent amino acids in that they have hydroxyl groups on their side chains they are found only in a few connective-tissue proteins, such as collagen. Thyroxine differs from tyrosine in that it has an extra iodine-containing aromatic group on the side... 

Fig. 4.4. Resolution of basic amino acids as a function of pH. Chromatographic conditions column, Dowex 50,17.5 jim (125 X 0.636 cm) mobile phase, 0.267 M citrate ion, 0.8 M sodium ion flow rate, 0.5 ml/min temperature, 60 °C detection, ninhydrin derivatives at 540 nm and 440 nm. 1, jS-aminoisobutyric acid 2, y-aminobutyric acid 3, ethanolamine 4, ammonia 5, hydroxylysine 6, ornithine 7, lysine 8, 1-methyl-histidine 9, histidine 10, 3-methylhistidine 11, anserine 12, 2-amino-3-guanidinopropionic acid 13, canavanine 14, tryptophan 15, camosine 16, arginine. Reproduced from Hamilton (1963), with permission.

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